Antihypertensive drug concentration measurement combined with personalized feedback in resistant hypertension: a randomized controlled trial.

BACKGROUND: Adherence to antihypertensive drugs (AHDs) is crucial for controlling blood pressure (BP). We aimed to determine the effectiveness of measuring AHD concentrations using a dried blood spot (DBS) sampling method to identify nonadherence, combined with personalized feedback, in reducing resistant hypertension. METHODS: We conducted a multicenter, randomized, controlled trial (RHYME-RCT, ICTRP NTR6914) in patients with established resistant hypertension. Patients were randomized to receive either an intervention with standard of care (SoC) or SoC alone. SoC consisted of BP measurement and DBS sampling at baseline, 3 months (t3), 6 months (t6), and 12 months (t12); AHD concentrations were measured but not reported in this arm. In the intervention arm, results on AHD concentrations were discussed during a personalized feedback conversation at baseline and t3. Study endpoints included the proportion of patients with RH and AHD adherence at t12. RESULTS: Forty-nine patients were randomized to receive the intervention+SoC, and 51 were randomized to receive SoC alone. The proportion of adherent patients improved from 70.0 to 92.5% in the intervention+SoC arm ( P  = 0.008, n  = 40) and remained the same in the SoC arm (71.4%, n  = 42). The difference in adherence between the arms was statistically significant ( P  = 0.014). The prevalence of resistant hypertension decreased to 75.0% in the intervention+SoC arm ( P  < 0.001, n  = 40) and 59.5% in the SoC arm ( P  < 0.001, n  = 42) at t12; the difference between the arms was statistically nonsignificant ( P  = 0.14). CONCLUSION: Personalized feedback conversations based on DBS-derived AHD concentrations improved AHD adherence but did not reduce the prevalence of RH.

Lack of efficacy of thrombopoietin and granulocyte-macrophage colony-stimulating factor after total body irradiation and autologous bone marrow transplantation in Rhesus monkeys.

OBJECTIVE: If administered in a sufficiently high dose to overcome receptor-mediated clearance and in a well-scheduled manner, thrombopoietin (TPO) prominently stimulates hematopoietic reconstitution following myelosuppressive treatment and potentiates the efficacy of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). However, TPO alone is not effective after bone marrow transplantation. Based on results of GM-CSF and TPO treatment after myelosuppression that resulted in augmented thrombocyte, reticulocyte, and leukocyte regeneration, we evaluated TPO/GM-CSF treatment after lethal irradiation followed by autologous bone marrow transplantation. MATERIALS AND METHODS: Young adult Rhesus monkeys were subjected to 8-Gy total body irradiation (TBI) (x-rays) followed by transplantation of 10(7)/kg unfractionated bone marrow cells. TPO 5 microg/kg was administered intravenously at day 0 to obtain rapidly high levels. Animals then were treated with 5 microg/kg Rhesus TPO and 25 microg/kg GM-CSF given SC on days 1 to 14 after TBI. RESULTS: The grafts shortened the profound pancytopenia induced by 8-Gy TBI from 5-6 weeks to 3 weeks. The combination of TPO and GM-CSF did not significantly influence the recovery patterns of thrombocytes (p = 0.39), reticulocytes (p = 0.08), white blood cells (p = 0.08), or bone marrow progenitors compared to TPO alone. CONCLUSIONS: The present study demonstrates that, after high-dose TBI and transplantation of a limited number of unfractionated bone marrow cells, simultaneous administration of TPO and GM-CSF after TBI is ineffective in preventing pancytopenia. This result contrasts sharply with the prominent stimulation observed in a 5-Gy TBI myelosuppression model, despite a similar level of pancytopenia in the 8-Gy model of the present study. The discordant results of this growth factor combination in these two models may imply codependence of the hematopoietic response to TPO and/or GM-CSF on other factors or cytokines.

Efficient detection and selection of immature rhesus monkey and human CD34+ hematopoietic cells expressing the enhanced green fluorescent protein (EGFP).

The feasibility of using the enhanced green fluorescent protein (EGFP) as a selectable reporter molecule of retroviral-mediated gene transfer in immature rhesus monkey and human CD34+ hematopoietic cells was examined. Retroviral transduction with the MFG-EGFP retroviral vector resulted in readily detectable EGFP expression in 27% of human and 11-35% of rhesus monkey bone marrow cells, and in 17-38% of rhesus monkey peripheral blood cells mobilized with FLT3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF). In addition, we used the human CD34+ KG1A cell line as a model to study viability and growth of successfully transduced cells. Cultures of mock- and EGFP-transduced KG1A cells generated equal viable cell numbers for at least 1 month, indicating the absence of a cytotoxic effect of EGFP expression in these cells. FACS selection on the basis of EGFP and CD34 expression resulted in enriched subsets (> or = 87%) of CD34+ EGFP-negative and CD34+ EGFP-positive KG1A, rhesus monkey and human bone marrow cells, demonstrating the potential of obtaining almost pure populations of transduced immature hematopoietic cells. EGFP expression was also readily demonstrated in erythroid and granulocyte/macrophage colonies derived from the CD34+ EGFP-positive rhesus monkey and human bone marrow cells by either inverted fluorescence microscopy or flow cytometry. Using four-color flow cytometry, EGFP expression could also be demonstrated in viable and phenotypically defined immature subpopulations of the CD34+ cells, ie those expressing little or no HLA-DR (rhesus monkey) or CD38 (human) antigens at the cell surface. These results demonstrate that EGFP is a very useful marker to monitor gene transfer efficiency in phenotypically defined immature rhesus monkey and human hematopoietic cell types and to select for these cells by multicolor flow cytometry prior to transplantation.

The efficacy of recombinant thrombopoietin in murine and nonhuman primate models for radiation-induced myelosuppression and stem cell transplantation.

Radiation-induced pancytopenia proved to be a suitable model system in mice and rhesus monkeys for studying thrombopoietin (TPO) target cell range and efficacy. TPO was highly effective in rhesus monkeys exposed to the mid-lethal dose of 5 Gy (300 kV x-rays) TBI, a model in which it alleviated thrombocytopenia, promoted red cell reconstitution, accelerated reconstitution of immature CD34+ bone marrow cells, and potentiated the response to growth factors such as GM-CSF and G-CSF. In contrast to the results in the 5 Gy TBI model, TPO was ineffective following transplantation of limited numbers of autologous bone marrow or highly purified stem cells in monkeys conditioned with 8 Gy TBI. In the 5 Gy model, a single dose of TPO augmented by GM-CSF 24 h after TBI was effective in preventing thrombocytopenia. The strong erythropoietic stimulation may result in iron depletion, and TPO treatment should be accompanied by monitoring of iron status. This preclinical evaluation thus identified TPO as a potential major therapeutic agent for counteracting radiation-induced pancytopenia and demonstrated pronounced stimulatory effects on the reconstitution of immature CD34+ hemopoietic cells with multilineage potential. The latter observation explains the potentiation of the hematopoietic responses to G-CSF and GM-CSF when administered concomitantly. It also predicts the effective use of TPO to accelerate reconstitution of immature hematopoietic cells as well as possible synergistic effects in vivo with various other growth factors acting on immature stem cells and their direct lineage-committed progeny. The finding that a single dose of TPO might be sufficient for a clinically significant response emphasizes its potency and is of practical relevance. The heterogeneity of the TPO response encountered in the various models used for evaluation points to multiple mechanisms operating on the TPO response and heterogeneity of its target cells. Mechanistic mouse studies made apparent that the response of multilineage cells shortly after TBI to a single administration of TPO is quantitatively more important for optimal efficacy than the lineage-restricted response obtained at later intervals after TBI and emphasized the importance of a relatively high dose of TPO to overcome initial c-mpl-mediated clearance. Further elucidation of mechanisms determining efficacy might very well result in a further improvement, e.g., following transplantation of limited numbers of stem cells. Adverse effects of TPO administration to myelosuppressed or stem cell transplanted experimental animals were not observed.

In vivo expansion of hemopoietic stem cells.

Under conditions of steady-state hemopoiesis, a small fraction of immature hemopoietic cells, including stem cells, circulates in peripheral blood (PB). In rhesus monkeys, a median number of 1.2 x 10(7)/l CD34+ cells was observed as opposed to a median number of 1.5 x 10(9)/l in aspirated bone marrow (BM). The concentration of circulating CD34+ cells is therefore approximately two logs less than that in BM. Since a 4-kg rhesus monkey has an estimated number of 3 x 10(10) BM cells and approximately 300 ml of blood, the fraction of CD34+ cells that circulates can be estimated at approximately 0.4% of the total pool of CD34+ cells. During hemopoietic reconstitution following a cytotoxic insult such as results from a midlethal dose of TBI, PB CD34+ cell numbers appeared to be correlated to those of BM, suggesting that PB CD34+ cells may reflect reconstitution of BM CD34+ cells. Reconstitution of BM immature cells can be accelerated by treatment with pharmacological doses of growth factors, resulting in largely expanded immature cell populations within a few weeks after TBI. Growth factors observed to exert such an effect included, notably, thrombopoietin. Such an acceleration can be monitored by daily assessment of circulating CD34+ cells. Expansion of immature circulating cells indicates expansion of similar cells in the bone marrow rather than growth factor-induced selective mobilization of immature cells.

The efficacy of single-dose administration of thrombopoietin with coadministration of either granulocyte/macrophage or granulocyte colony-stimulating factor in myelosuppressed rhesus monkeys.

Thrombopoietin (TPO) was evaluated for efficacy in a placebo-controlled study in rhesus monkeys with concurrent administration of either granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF, (G-CSF). Rhesus monkeys were subjected to 5 Gy total-body irradiation (TBI), resulting in 3 weeks of profound pancytopenia, and received either TPO 5 microg/kg intravenously (I.V.) at day 1 (n = 4), GM-CSF 25 microg/kg subcutaneously (S.C.) for 14 days (n = 4), TPO and GM-CSF (n = 4), G-CSF 10 microg/kg/d S.C. for 14 days (n = 3), TPO and G-CSF (n = 4), or placebo (carrier, n = 4; historical controls, n = 8). Single-dose I.V. treatment with TPO 1 day after TBI effectively counteracted the need for thrombocyte transfusions (provided whenever thrombocyte levels were <40 x 10(9)/L) and accelerated platelet reconstitution to normal levels 2 weeks earlier than placebo controls. TPO/GM-CSF was more effective than single-dose TPO alone in stimulating thrombocyte regeneration, with a less profound nadir and a further accelerated recovery to normal thrombocyte counts, as well as a slight overshoot to supranormal levels of thrombocytes. Monkeys treated with TPO/GM-CSF uniformly did not require thrombocyte transfusions, whereas those treated with GM-CSF alone needed two to three transfusions, similar to the placebo-treated monkeys, which required, on average, three transfusions. Also, reticulocyte production was stimulated by TPO and further augmented in monkeys treated with TPO/GM-CSF. TPO alone did not stimulate neutrophil regeneration, whereas GM-CSF shortened the period of neutrophil counts less than 0.5 x 10(9)/L by approximately 1 week; TPO/GM-CSF treatment elevated the neutrophil nadir, but did not further accelerate recovery to normal values. TPO also augemented the neturophil response to G-CSF, resulting in similar patterns of reconstitution following TPO/G-CSF and TPO/GM-CSF treatment. TPO/GM-CSF resulted in significantly increased reconstitution of CD34+ bone marrow cells and progenitor cells such as GM-CFU and BFU-E. Adverse effects of combining TPO with the CSFs were not observed. It is concluded that (1) a single I.V. administration of TPO is sufficient to prevent severe thrombocytopenia following myelosuppression, (2) TPO/G-CSF and TPO/GM-CSF treatment result in distinct response patterns, with TPO/GM-CSF being superior to TPO/G-CSF in stimulating thrombocyte and erythrocyte recovery while being equivalent in stimulating neutrophil recovery; and (3) TPO significantly improves the performance of CSFs in alleviating severe neutropenia.